Depression is among the most debilitating psychiatric disorders, yet few pharmacological approaches exist for treating depression. Tricyclic antidepressants, which inhibit serotonin and noradrenaline reuptake transporters, and monoamine oxidase inhibitors, which inhibit the major catabolic enzyme for monoamine neurotransmitters, have been available for over 50 years. Another treatment strategy that has been utilized for many years is electroconvulsive seizure therapy. More recently, selective serotonin reuptake inhibitors have been developed. Nevertheless, today's treatments are sub-optimal, with only approximately 50% of all patients demonstrating complete remission, although more (up to 80%) show partial responses (Nestler et al., 2002; Neuron 34, 13-25). There therefore exists an urgent need for novel strategies for the treatment of depression.
Serotonergic pathways in the brain have been well-established as critical for regulation of mood and depletion of serotonin in brain cells has been associated with production of behavioral depression (Cooper et al. 1991. The Biochemical Basis of Neuropharmacology, 6th Edition. Oxford university Press: New York, pages 338-366). As a result, pharmacological research has focused on development of compounds that alter serotonergic function as a way to treat depression. Serotonin (also known as 5-HT or 5HT) acts on neuronal tissue to elicit its responses through the actions of specific receptors. Serotonin released from a presynaptic neuron acts at specific receptors on the postsynaptic neuron. Most of the current treatment paradigms for depression involves modulation of serotonergic neurotransmission through the usage of agents that alter the level of serotonin in the synaptic cleft.
Fifteen types of serotonin receptors have been identified in mammalian CNS tissue, cloned and pharmacologically characterized (Barnes et al., 1999, Neuropharmacology 38, 1083-1152). They are divided into seven different subclasses: 5-HT1A-F, 5-HT2A-C, 5-HT3A-B, 5-HT4, 5-HT5, 5-HT6 and 5-HT7 receptors. All of them are metabotropic receptors, with the exception of 5-HT3 receptors, which are ionotropic. These serotonin receptors act via the following second messenger systems: 5-HT1-class receptors and 5-HT5A and B receptors decrease cAMP formation; 5-HT2-class receptors increase inositol triphosphate and diacylglycerol formation; 5-HT3 receptors increase Na+ and Ca2+ influx; and 5-HT4, 5-HT6, and 5-HT7 receptors increase cAMP formation. Thus, serotonin can activate multiple second messenger systems, and as a consequence, the action of most anti-depressants is rather non-specific.
So far, there are no specific agonist or antagonists that act at any of the serotonin receptors that have shown a better therapeutic profile than the currently employed anti-depressants. This indicates that an altered activity at multiple serotonin receptors may be needed for anti-depressants to achieve their effects and that the antidepressant properties are achieved by the integration of several neurotransmitter-mediated intracellular signaling cascades within the postsynaptic neuron.
Proteins that integrate the effects of multiple serotonin receptors may therefore be involved in the anti-depressant efficacy of tricyclic antidepressants, selective serotonin reuptake inhibitors and/or monoamine oxidase inhibitors. One such protein, DARPP-32, (Dopamine- and cAMP-regulated phosphoprotein, Mr 32,000) was discovered as a major target for dopamine and cAMP in the brain (Walaas et al. 1983. Nature 301:69-71). DARPP-32 is enriched in the two major projection areas for dopaminergic neurons, the prefrontal cortex and the striatum. Prefrontal cortex and the striatum receive a moderate to high serotonergic innervation (Steinbusch, 1981, Neuroscience 6, 557-618), which is further increased when the levels of dopamine in this region are decreased (Rozas et al., 1998, Neurosci. Lett. 245, 151-154). Several serotonin receptors, most notably 5-HT1B, 5-HT1E, 5-HT2A, 5-HT2C, 5-HT3, 5-HT4, and 5-HT6, are expressed in striatum (Barnes et al., 1999, Neuropharmacology 38, 1083-1152).
Over the past years it has been established that DARPP-32 plays an obligatory role in the biochemical, electrophysiological, transcriptional, and behavioral effects of dopamine (Greengard, P. et al. 1999. Neuron 23:435-447). Dopamine, via D1 receptor-mediated activation of protein kinase A (PKA), phosphorylates DARPP-32 at Thr34 and thereby converts DARPP-32 into a potent inhibitor of protein phosphatase-1 (PP-1) (Walaas et al. 1983. Nature 301:69-71; Hemmings et al. 1984. Nature 310:503-505). Phospho-DARPP-32 is dephosphorylated by PP2B (also termed calcineurin). Additional kinases and phosphatases have been shown to phosphorylate DARPP-32 at distinct sites and to regulate its function.
Casein Kinase 1 (CK1) was one of the first serine/threonine protein kinases to be isolated and characterized (Gross, S. D. and R. A. Anderson. 1998. Cell Signal 10:699-671). It is a ubiquitous enzyme that can be found in the nucleus and the cytosol of cells and bound to the cytoskeleton or to cell membranes. In the central nervous system (CNS), CK1 appears to play a role in regulation of circadian rhythm (Camacho, F. et al. 2001. FEBS Lett. 489:159-165) and intracellular trafficking (Murakami, A. et al. 1999. J. Biol. Chem. 274:3804-3810; Panek, H. R. et al. 1997. EMBO J. 16:4194-4204; Wang, X. et al. 1996. Mol. Cell. Biol. 16:5375-5385). In the neostriatum, CK1 has been found to phosphorylate DARPP-32 at Ser137 (Desdouits, F. et al. 1995. J. Biol. Chem. 270:8772-8778). Phosphorylation of Ser137 influences the ability of phospho-Thr34 to be dephosphorylated by PP2B (Desdouits, F. et al. 1995. Proc. Natl. Acad. Sci. USA 92:2682-2685; Desdouits, F. et al. 1998. Biochem. J. 330:211-216). PP2C is also involved in this complex and in regulating the state of DARPP-32 phosphorylation at Thr34, since PP2C decreases phosphorylation of DARPP-32 at the Ser137 site (Desdouits et al. 1998. Biochem. J. 330:211-216).
In addition to PP2B, PP2C and CK1, cyclin-dependent kinase 5 (cdk5) and PP2A are also involved in regulating the state of phosphorylation of DARPP-32. Cdk5 was originally identified as a homologue of p34cdc2 protein kinase. Subsequent studies have shown that unlike cdc2, cdk5 kinase activity is not detected in dividing cells. Instead, the active form of cdk5 is present only in differentiated neurons, where it associates with a neuron-specific 35 kDa regulatory subunit, termed p35. Cdk5/p35 plays a variety of roles in the developing and adult nervous system. Recent studies have linked mis-regulation of cdk5 to Alzheimer's disease (Kusakawa, G. et al. 2000. J. Biol. Chem. 275:17166-17172; Lee, M. S. et al. 2000. Nature 405:360-364; Nath, R. et al. 2000. Biochem. Biophys. Res. Commun. 274:16-21; Patrick, G. N. et al. 1999. Nature 402:615-622). In these studies, conversion of p35 to p25 by the action of calpain causes prolonged activation and altered localization of cdk5. In turn, cdk5/p25 can hyperphosphorylate tau, disrupt cytoskeletal structure and promote apoptosis of primary neurons.
Recent studies have also shown that cdk5 phosphorylates DARPP-32 at Thr75 (Nishi, A. et al. 2000. Proc. Natl. Acad. Sci. USA 97:12840-12845; Bibb, J. A. et al. 1999. Nature 402:669-671). DARPP-32 phosphorylated at Thr75 is an inhibitor of PKA (Bibb et al. 1999. Nature 402:669-671). Phosphorylation of DARPP-32 at Thr75 by cdk5, by inhibiting PKA, decreases phosphorylation of Thr34 in DARPP-32 by PKA and plays an important modulatory role in the DARPP-32/PP1 cascade (Bibb, J. A. et al. 1999. Nature 402:669-671). Phospho-Thr75 is dephosphorylated by PP2A. The available evidence indicates that dopamine decreases phosphorylation of DARPP-32 at Thr75 by activating PKA which, in turn, activates PP2A and thereby increases the efficacy of the DARPP-32/PP1 signaling cascade (Nishi et al. 2000. Proc. Natl. Acad. Sci. USA 97:12840-12845).
Another study has linked the total levels of DARPP-32 with the pharmacological activity of certain anti-depressant compounds (Guitart, X. and E. J. Nestler. 1992. J. Neurochem. 59:1164-1167). These researchers demonstrated that chronic administration of lithium, imipramine, and tranylcypromine in rats produced significant increases in frontal cortex levels of DARPP-32 immunoreactivity, while administration of haloperidol, morphine, and cocaine were without effects on DARPP-32 immunoreactivity. Lithium is used for treatment of manic-depressive illness, while imipramine and tranylcypromine are anti-depressants. Imipramine acts by inhibiting norepinephrine re-uptake while tranylcypromine is a monoamine oxidase inhibitor.
Although research has focused on specific agonists and antagonists for these receptors and the way that serotonergic function is then modulated by these agonists and antagonists, little is known about the variety of intracellular signaling pathways linked to serotonin activity or about drugs that may be used to treat disorders related to the function of serotonergic intracellular signaling pathways. Therefore, there is an urgent need to provide new drug assays that can be used to develop novel drugs that can be used to treat disorders related to function of serotonergic intracellular signaling pathways.